ABSTRACT
Aim To study the therapeutic effect of CpG-ODN, an agonist of Toll-like receptor 9 (TLR9), on hypoxic/ischemic encephapathy in neonatal rats and investigate the mechanisms.Methods Fifty healthy 7-day-old neonatal Wistar rats (in either gender, weighing 12~17g) were randomly divided into sham operation group, HIBD group, and CpG-ODN low group(0.35 mL·kg-1), CpG-ODN middle group(1.40 mL·kg-1), CpG-ODN high group(5.60 mL·kg-1).The neurological function was scored after 48h operation;ten rats of each group was executed respectively and brains tissue was taken;HE staining was used to observe the brain pathological changes.Western blot assay was used to detect the expressions of TLR9 and phosphor-p38 mitogen-activated protein kinases(p-p38 MAPK), and enzyme linked immunosorbent assay (ELISA) method was adopted to detect TNF-α expression.Results The CpG-ODN low, middle group were improved in impairment significantly compared with the HIBD group, and the brain pathological change was lessened, while the CpG-ODN high group was impaired significantly compared with the HIBD group (P<0.05), and brain pathological change was sharpened.Western blot showed the up-regulation in TLR9 and p-p38 MAPK and a significant increase of the expression of TNF-α in the brain tissue in CpG-ODN group with statistical difference in HIBD group and sham operation group(P<0.05).Conclusions The neuro-behavioral score and nervous system function can be improved and the hypoxic/ischemic brain damage can be reduced in neonatal rats in the CpG-ODN low, middle group.The protective mechanisms may be suitably via activating p38 MAPK signaling pathway to promote p38 MAPK phosphory1ation and up-regulation of the expression of TNF-α in the brain tissue of rats.
ABSTRACT
Objective To investigate the feasibility of using clustered regularly interspaced short palindromic repeats ( CRISPR)-mediated genome editing to downregulate the expression of programmed cell death protein 1 (PD-1) on primary T cells by using a lentivirus delivery system. Methods Lentivirus vec-tors pLentiCRISPR A1-A6 containing different PD-1 genomic DNA sequences as single guide RNA ( sgRNA) for Cas9 targeting were constructed individually. The lentivirus vectors were tranduced into primary CD4 T cells. Flow cytometry analysis was performed to detect the expression of PD-1 for evaluating the knockout ef-ficiency. Results The lentivirus vectors pLentiCRISPR A1-A6 carrying six different target sites were con-structed and respectively tranduced into primary CD4 T cells. The expression of PD-1 accompanied with the activation of T cells. Co-expression of CD25 and PD-1 was observed on activated T cells. All of the six sites could be targeted by Cas9, of which A2 and A6 sites were more efficient in knocking out the gene encoding PD-1 with a rate of 19% and 29%, respectively. Conclusion This study suggests that it is feasible to knock out the expression of PD-1 on primary T cells by using CRISPR.